Prochlorococcus marinus responses to light and oxygen

¹ Department of Biology, Mount Allison University, 63B York St., Sackville NB, Canada, E4L 1G7.
² Department of Biology, University of British Columbia
³ Department of Chemistry, St. Frances Xavier University
⁴ Institute of Microbiology, Center Algatech, Laboratory of Photosynthesis, Novohradska 237, Trebon, CZ 37981, Czech Republic.
⁵ Institute of Oceanography, University of Gdansk, 46 Piłsudskiego St, Gdynia, Poland

Abstract

Introduction

Prochlorococcus marinus diversity

Prochlorococcus, a genus of Cyanobacteria, is the smallest known photosynthetic prokaryote, with cell diameters ranging from 0.5 to 0.7 µm [1]. Despite small cell size, P. marinus contribute 13 to 48% of net primary production in oligotrophic oceans, corresponding to about 30% of global oxygen production [2]. Prochlorococcus marinus growth is currently limited to between latitudes of 40°N to 40°S in open ocean waters, from surface to 300 m depth, thus spanning 3 orders of magnitudes of light irradiance [1,2].

Prochlorococcus marinus comprises many strains, organized into clades, defined by 16S-23S intergenic transcribed ribosomal sequence signatures [3]. The clades inhabit distinct ecological niches [4], originally defined as High-Light (HL) or Low-Light (LL). Only 5 out of 12 known Prochlorococcus genetic clades have cultured representatives to date; HLI, HLII, LLI, LLII/III and LIV [5]. Current niches of P. marinus strains span ocean water columns [2,6,7] and extend into regions with low dissolved oxygen concentrations [8–12].

Low-Light clades thrive in deeper ocean waters, extending beyond 200 m in depth [2], where only ~1% of the surface irradiance penetrates, primarily in the blue (450 nm) to green (520 nm) spectral range [13]. Clade LLI includes cultured strain NATL2A, which prefers moderate irradiances typical of between 30 and 100 m depth. Clades LLII and LLIII, including cultured strain SS120, are grouped together as the second oldest phylogenetic lineage diversifying in the P. marinus radiation, with an affinity for low light. Clade LLIV, including cultured strain MIT9313, falls near the base of the Prochlorococcus radiation, and is characterized by preference for low light, typical of depths from 120 m to 200 m [2]. LLIV members are, as yet, the only cultured strains to have been found in Oxygen Minimum Zones (OMZ). Some, as yet, uncultured P. marinus strains in clades LLV and LLVI also thrive in OMZ of the subtropical Atlantic and Pacific Oceans, where dissolved oxygen concentrations [O₂] can be less than 20 µM [9–12,14]. Prochlorococcus marinus LL ecotypes may indeed dominate the phytoplankton within OMZ [8,10,11], where they may be net O₂ consumers [15].

HL clades are more recently branching lineages, with reduced genome sizes in comparison to LL clades. High-Light clades are typically dominant picophytoplankters in near-surface, oligotrophic waters, characterized by high light levels and subdivided into clades based on iron adaptation [16–18]. Clade HLI, represented by cultured strain MED4, is adapted to high iron, and low temperatures, and originated from 5 m depth in the Mediterranean Sea [2]. Clade HLII, adapted to high iron, and high temperatures, is the most abundant P. marinus clade in the North Atlantic and North Pacific Oceans, often constituting over 90% of the total population [2], and are most numerous around 50 m depth [2]. Clade HLIII/IV is adapted to low iron [16–18].

Prochlorococcus marinus clades are nonetheless found in environments beyond their optimal habitats. HL clades inhabit depths overlapping with LL ecotypes [17,19,westNichePartitioningProchlorococcusPopulationsStratified1999?], while LL clades can occupy regions in OMZ at depths shallower than 40 m [10], exploiting ambient light levels above what LL clades were thought to tolerate.

Prochlorococcus and changing niches

Our changing climate is rapidly altering conditions for these specialized clades of marine picophytoplankton. Predictions indicate a net global increase of P. marinus cell abundances of 29% [20], along with poleward latitudinal shifts of at least 10° in marine phytoplankton niches by the end of this century [21] in response to warming waters, with increases in P. marinus of approximately 50% in the more poleward regions of their distributions.

Near the equator, photoperiod remains nearly constant at the ocean surface, approximately 12 hours (h) of daylight and 12 h of darkness throughout the year. The effective length of the photoperiod does, however, attenuate with depth as dawn and dusk light at depth drops below levels needed for biological processes. As P. marinus potentially expands its temperature-permissive niches poleward into temperate regions [20,21], it will encounter more pronounced seasonal variations in photoperiod regimes both at surface and at depth, with potentially complex effects upon viable growth niches [22,23]. For example, Vaulot et al. [24] showed that Prochlorococcus replication of DNA occurs in the afternoon, while cell division occurs at night. To our knowledge, no study has as yet addressed P. marinus growth responses in relation to a range of photoperiods.

Climate change is also rapidly changing ocean chemistry. By the end of this century, surface ocean pH is projected to decline by 0.1 to 0.4 due to projected increases in carbon dioxide concentrations [25]. Moreover, substantial changes in the global water cycle, leading to extensive changes in worldwide precipitation patterns, are affecting ocean salinity levels on a global scale, and ice melts due to rising temperatures are impacting salinity levels in the Arctic and Northwest Atlantic oceans [26]. Increasing sea temperatures are also causing decreases in [O₂] across global oceans [27], particularly toward the poles [28]. Warmer ocean waters decrease oxygen solubility at the surface, and increase stratification, which in turn decreases oxygen mixing downwards by ocean currents [25]. Models predict that OMZ in the Pacific and Indian Oceans are expanding [25,29], although the cores of the OMZ, where the oxygen levels are lowest, may actually contract [29].

We used the OceansMap Protein Portal (OPP; https://proteinportal.whoi.edu/) [30] to analyze the distribution of proteins from clades of P. marinus in samples taken across a range of [O₂] and depth, which in turn correlates to peak light at the site of sampling. In parallel we analyzed the growth and physiological responses of representative strains from three clades of P. marinus under a matrix of [O₂], light levels, spectral waveband ranges, and photoperiods, to approximate eco-physiological conditions representative of current and hypothetical future ocean zones. Prochlorococcus marinus MED4, a clade HLI strain, was isolated near the ocean surface (5m depth) of the Mediterranean Sea where [O₂] is near saturation, light levels are high and spectral bias from full solar irradiance is minimal. Prochlorococcus marinus SS120, a clade LLII/III strain, was isolated from the Sargasso Sea at a depth of 120 m, while P. marinus MIT9313, a clade LLIV strain, was isolated from the North Atlantic Gulf Stream at a depth of 135 m [31]. At these depths, light attenuation and spectral shifts occur, resulting in low blue light, while [O₂] varies from near-surface saturation levels to decreased concentrations, but does not necessarily decrease systematically with depth [32].

Photosynthetic organisms absorb light energy within the Photosynthetically Active Radiation (PAR) range, 350 to 700 nm, for photosynthesis [33]. Photosynthetically Usable Radiation (PUR) represents the fraction of PAR that is absorbed by the pigments of a given photosynthetic organism [33], taking into account the specific spectral wavebands these pigments absorb. Prochlorococcus marinus Pcb light-harvesting complexes show an absorption maxima of 442 nm for divinyl chlorophyll a and 478 nm for divinyl chlorophyll b [34] allowing P. marinus to efficiently harvest blue light in the 400 nm to 500 nm range [33] corresponding to blue spectral wavelengths prevailing in deep ocean habitats [13]. In P. marinus small cell diameters, from 0.5 to 0.7 µm [1], and simple cell structures, minimize the complication of pigment package effect or intracellular self-shading [35] contributing to efficient optical absorption, although photosynthetic efficiency may vary among clades [31,36].

Given the different spectral light regimes typical of the niches of different ecotypes, expressing growth rates in terms of cumulative diel PUR might simplify different photoperiods, spectral bands, and PAR levels into a common parameter, making growth response comparisons across strains and different oxygen levels more accessible. We aimed to detect whether growth responses are driven simply by cumulative diel PUR, or whether specific photoperiods, spectral bands or PAR levels have independent, albeit interacting, effects on growth. We therefore analyzed growth rates in terms of cumulative diel PUR.

We discuss our findings in relation to analyses of genomic sequences [37] across clades of P. marinus, showing that differences in the expression and presence of genes encoding protein turnover, oxygen-dependent enzymes, and DNA repair enzymes, can explain the differential growth responses of strains under the matrix of light and [O₂] conditions of this study.

Materials and methods

MetaProteomics

The OceansMap Protein Portal is an open access online data repository (Woods Hole Oceanographic Institute, WHOI) of mass spectroscopy data on marine microbial peptides, sampled from various depths and locations worldwide [30]. We screened a subset of the OPP for proteins annotated as from Prochlorococcus strains, to identify differential strategies employed by strains living at varying depths and oxygen levels within the marine water column. We focused on proteins mediating photosynthesis and protein metabolism from depths of 20 to 200 m below the ocean surface. The samples for metaproteomic analyses were collected from 12 locations in the tropical North Pacific ocean along 150 W from 18 N of the equator between October 1, 2011 and October 25, 2011 during the voyage of the R/V Kilo Moana MetZyme cruise KM1128 (https://www.rvdata.us/search/cruise/KM1128; original datasets in the Biological and Chemical Oceanography Data Management Office repository; https://www.bco-dmo.org/project/2236). Oxygen concentration levels at the location of sampling were recorded. The methodology for sample collection and peptide analysis are described by Saito et al. [38,39].

MetaProteomics bioinformatic analyses

Metaproteomic datasets were obtained from the KM1128 entry in the BCO-DMO database (https://www.bco-dmo.org/deployment/59053) accessed via the OPP in June 2019. Datasets contained: i) Peptide sequences and sample identification (ID) number; ii) Sample ID number, station, depth in meters below the surface the sample was collected at, best-hit BLASTP protein and species annotation and the corresponding Uniprot Entry number for the identified proteins; iii) Sample station depth and [O₂].
The depth and [O₂] were joined to peptide sequence and BLASTP annotations by ID number, depth and station using tidyverse package [40] running under R v4.1.3 and RStudio v2023.06.0 [41]. The resulting merged dataset was filtered for those Prochlorococcus peptides, detected from 0 to 300 m below the surface, annotated as a subunit of Prochlorococcus chlorophyll binding proteins (Pcb); Photosystem II (PSII); Cytochrome b₆f (Cytb₆f); Photosystem I (PSI); NADPH Dehydrogenase (NDH); Plastoquinol Terminal Oxidase (PTOX); Plastocyanin (PC); Ferredoxin (Fd); Ribulose-1,5-bisphosphate oxygenase (RUBISCO); ATP Synthase; FtsH proteases (FtsH) or ribosomes. Detected peptides were re-annotated for consistency and labelled, where feasible, according to strain, clade, subunit and protein complex. Full protein sequences corresponding to detected proteins were obtained from UniProt (https://www.uniprot.org/) and analyzed in Molecular Evolution and Genetic Analyses X (MEGAX) software (https://www.megasoftware.net/). Sequences for proteins for each of the thirteen Prochlorococcus strains identified in the dataset were aligned with MUSCLE using UPGMA cluster method and a lambda of 24 with a -2.9 gap open penalty and 1.20 hydrophobicity multiplier. Overall mean pairwise distance between protein sequences was determined using bootstrap variance estimation methods. Maximum likelihood phylogenetic trees were assembled using 1000 bootstrap replications with a 95% site coverage cut off. Prochlorococcus FtsH isoform identities, and functions, were inferred by sequence comparisons to the characterized four isoforms of FtsH protease of Synechocystis sp. PCC6803 [42]. Data for each strain was plotted against depth and [O₂] and sampling station.

When assessing the presence of a particular protein complex at a sampling location, all peptides belonging to all subunits of the complex were included to give the greatest number of data points. As this data was acquired on a discovery mission rather than through targeted peptide approaches, it is difficult to discern accuracies of strain assignment annotations, particularly as the proteins of interest in this study are highly conserved across strains. We are, however, confident in clade classifications for each protein examined. A caveat to interpretation of this data is the peptide detection bias inherent to mass spectrometry XXX Aurora CITATION XXX. The data is also limited by the number and nature of protein spectra in the SEQUEST database: a peptide sequence was not determined unless there is already a known spectrum for that peptide in the SEQUEST database, hence some peptides of interest may not be identifiable. Furthermore, a peptide must be detected above a certain threshold abundance in order to be considered an accurate ‘hit’.

Prochlorococcus culturing and experimental design

Three xenic cultures of P. marinus were obtained from Bigelow Labs, NCMA Maine, USA. MED4 (CCMP1986) is from High-Light adapted (HLI) clade; SS120 (CCMP1375) is from Low-Light adapted (LLII/III) clade; and MIT9313 (CCMP2773) is from Low-Light adapted (LLIV) clade. Cultures were maintained in incubators set to 22°C with an on/off light/dark cycle of 12 h. The PAR level for maintenance cultures reflected PAR in the source niche of the ecotype; MED4, of 160 µmol photons m^-2 s^-1 with illumination from STANDARD Products Inc. Cool White F24T5/41K/8/HO/PS/G5/STD, 24 watts, fluorescent bulbs; SS120 and MIT9313 at 30 µmol photons m^-2 s^-1 with illumination from Philips Cool White F14T5/841 Alto, 14 watts, fluorescent bulbs. To maintain active growth all strains were transferred weekly with 1 in 5 dilutions with Pro99 media [43] prepared with autoclaved artificial seawater [44].

Controlled growth experiments were performed using MCMIX-OD or MC1000-OD PSI Multicultivators (Figure 13; PSI, Drásov, Czech Republic). Each multicultivator individually controls 8 tubes at a common temperature of 22°C. Each tube containing 70 mL of Pro99 media was inoculated with 10 mL of growing maintenance culture. In a factorial matrix design, each tube was then subject to an individual combination of sinusoidal photoperiod (4, 8, 12, 16 h); reaching a peak PAR (30, 90, 180 µmol photons m^-2 s^-1), with defined spectral bandwidth (White LED, 660 nm, 450 nm). [O₂] levels (2.5 µM, 25 µM, 250 µM) were imposed by bubbling tubes with varying ratios of air and Nitrogen (N₂), with consistent 0.05% of Carbon Dioxide (CO₂) gas, delivered through a 0.2 μm sterile microfilter via a G400 gas mixing system (Qubit Systems Inc., Kingston, Ontario, Canada). [O₂] in situ was verified using oxygen optodes (PyroScience, Germany) inserted into tubes for real-time measurements, with a temperature probe in the bath of the bioreactor to correct [O₂] measures for temperature fluctuations. In addition, the Pyroscience software corrected [O₂] based on the salinity of the media (32 ppt). The flow rate of the gas mixture was controlled, but variations in bubbling speed, PAR and culture density affected the [O₂] achieved in each tube. A low [O₂] of 0.5 µM - 5 µM (reported as 2.5 µM hereafter), was achieved by sparging with a gas mixture containing 99.95% N₂ and 0.05% CO₂. An intermediate [O₂] of 10 - 25 µM (reported hereafter as 25 µM) was achieved by sparging with a gas mixture containing 98.95% N₂, 0.05% CO₂ and 1% O₂. A high O₂ of 200 µM - 280 µM (reported hereafter as 250 µM) was achieved by sparging with lab air (78% N₂, 21% O₂, 1% Ar and 0.05% CO₂).

The full crossing of all factor levels would yield 4 x 3 x 3 x 3 = 108 treatments, x 3 strains for 324 possible combinations. Consistent absence of growth of some strains under some levels of photoperiod, PAR, or [O₂] meant we completed 268 growth factor treatment combinations.

In situ measurements of Optical Density (OD) 680 nm, a proxy for cell suspension density, cell size dependent scatter and cell chlorophyll content; and OD 720 nm, a proxy for cell suspension density and cell size dependent scatter, were recorded every 5 minutes over least 8 to 14 days, depending on the duration of the lag phase, if any.

PAR of 180, 90 or 30 µmol photons m^-2 s^-1, and spectral wavebands (white LED full spectrum, 660 nm (red light), and 450 nm (blue light)) were chosen to approximate light levels and spectral colours spanning the vertical ocean water column, from near-surface to the lower euphotic zone depths. Photoperiods were chosen to approximate diel cycles characteristic of current and hypothetical future niches of P. marinus; 16 h represents temperate (45°N) summer at the ocean surface; 12 h for equatorial (0°N) ocean surface or temperate (45°N) spring and fall ocean surface or temperate (45°N) summer at deeper ocean depths; 8 h for temperate (45°N) winter at the surface or at temperate (45°N) spring and fall at depth and equatorial (0°N) deep ocean depths; and 4 h for temperate (45°N) winter or deep ocean depths during temperate (45°N) spring and fall.

Growth rate analysis

Data files (.csv) saved from the Multicultivator software were imported into R-Studio for data management [40], growth rate calculations, comparisons of model fits [45], and visualization. The chlorophyll proxy optical density (OD₆₈₀ - OD₇₂₀; ΔOD) was used to determine the chlorophyll specific growth rate (µ, d^-1) for each treatment combination. We first used a rolling mean from the R package zoo [46] to calculate the average ΔOD data over a 1-hour window to lower the influence of outlier points and remove data points collected during post stationary phase, when applicable. We used the Levenberg-Marquardt algorithm [47] modification of the non-linear least squares, using the R package minpack.lm [48], to fit a logistic equation (Equation (1)); where ΔOD_max is maximum ΔOD, ΔOD_min is minimum ΔOD, t is time duration over the growth trajectory.

\[\begin{equation} µ = \frac{ΔOD_{max} × ΔOD_{min} × exp^{(µ × t)}}{ΔOD_{max} + (ΔOD_{min} × exp^{((µ × t) - 1)})} \tag{1} \end{equation}\]

Figure 14 is an example of chlorophyll specific growth estimates fitted from the high resolution ΔOD measurements for each tube in a Multicultivator. The residuals of the logistic growth curve fit are shown and the growth spectral waveband is plotted and illustrates the imposed PAR (µmol photons m^-2 s^-1) and photoperiod (h).

A Generalized Additive Model (GAM) [49] was applied to the relation of chlorophyll-specific µ, d^-1 to photoperiod and PAR level, for each growth [O₂] level, and for the blue and red wavebands for growth, for each P. marinus strain in this study. The R package mgcv [50] was used to model the growth rate with smoothing terms and indicate the 90, 50 and 10% quantiles for growth rate across the levels of factors. Only growth rate estimates for which the amplitude of standard error was smaller than 30% of the fitted growth rate were included in the GAM. Our priority was the effects of ecologically relevant blue light on growth trends. We also included GAM analyses of growth responses to red light, which is not ecophysiologically relevant, but which might prove mechanistically informative [51].

Estimation of photosynthetically usable radiation

To estimate the Photosynthetic Usable Radiation (PUR), a proxy of incident photons absorbed by the cells, for each P. marinus ecotype, the imposed Photosynthetic Active Radiation (PAR) was first determined using the reported delivery of sinusoidal diel PAR regimes by the Multicultivators, point validated using a LI-250 quantum sensor (LI-COR Inc.,Lincoln, NE, USA). An emission profile from 400 nm to 700 nm of each coloured LED light of the MCMIX-OD Multicultivator and the white LED light of the MC1000-OD Multicultivator was obtained using a Jaz spectrometer (Ocean Optics, Inc.,Dunedin, FL, USA) equipped with a fiber optic cable, HH2 FiberOpticJmp (Part number A901073, Malvern Panalytical Ltd, Malvern, UK). Each LED spectrum was then normalized to its emission maximum. An in-vivo whole cell absorbance spectrum for each P. marinus strain under each spectral growth condition was obtained using the Olis 14 UV/VIS Clarity Spectrophotometer (Olis Inc., Bogart, GA, USA) to scan across range of λ = 350 nm to 750 nm at 1 nm intervals. The path length of the internally reflective cavity of the Olis spectrophotometer was corrected to a 1 cm path length using the Javorfi correction method [52] on PRO 99 media subtracted whole cell absorbance spectra. The blank-corrected whole cell absorbance spectra were normalized to the absorbance maximum of divinyl chlorophyll a (Chl a₂), determined for each spectra, falling between 400 nm and 460 nm.

An integrated weighting equation (2) [33] was used to determine the weighted PUR spectrum P(λ); where A(λ) is the blank subtracted, Chl a₂ peak normalized whole cell absorbance spectrum for each P. marinus ecotype, over 400 nm to 700 nm, A(λ); and E(λ) is the peak normalized emission spectrum of the imposed LED growth light, over 400 nm to 700 nm.

\[\begin{equation} P(λ) = A(λ) × E(λ) \tag{2} \end{equation}\]

Actinic PUR levels (µmol photons m^-2 s^-1) were calculated from actinic PAR (µmol photons m^-2 s^-1) levels using the equation (3) from [33]; where P(λ) is the weighted PUR absorbance spectrum from equation (2), E(λ) is the imposed growth light emission spectrum from equation (2) and PAR is the imposed peak light level (µmol photons m^-2 s^-1). Figure (1) shows the calculated absorbed peak PUR (µmol photons m^-2 d^-1) versus imposed actinic peak PAR (µmol photons m^-2 s^-1) for each strain and each spectral waveband (nm).

\[\begin{equation} PUR = \frac{\int_{400}^{700} P(λ)}{\int_{400}^{700} E(λ)} × PAR \tag{3} \end{equation}\]

The applied photoperiods were delivered using the sinusoidal circadian light function of the PSI Multicultivator to simulate light exposure approximating sun rise through to sunset. The area under the sinusoidal curves is equivalent to the area of a triangular photoregime of equivalent photoperiod (Campbell, unpub), therefore the equation to determine the cumulative diel PUR (µmol photons m^-2 d^-1) is one half of the base (photoperiod) multiplied by the height (PUR) (Equation (4)); where PUR is the actinic absorbed light calculated from equation (3) (µmol photons m^-2 s^-1), 3600 is the time conversion from seconds to hour and photoperiod is the imposed photoperiod (h).

\[\begin{equation} Cumulative~diel~PUR = \frac{PUR × 3600 × Photoperiod}{2} \tag{4} \end{equation}\]

Figure 15 provide visual representations of PUR, the black solid line and shaded area, in relation to the imposed PAR, the dotted line, under each imposed spectral wavebands for P. marinus MED4 (A,B,C), SS120 (D,E,F) and MIT9313 (G,H,I). Figure (1) shows the relationship between calculated PUR versus imposed PAR for each P. marinus and each spectral waveband.

We fit the response of chlorophyll specific growth rate to cumulative diel PUR using the equation of Harrison and Platt [45] across strain and [O₂] to compare growth response between 660 nm (red) and 450 nm (blue) growth light and additionally, to compare growth response between specific photoperiods (4 h, 8 h, 12 h, 16 h) and a fit across pooled photoperiod data. To examine statistical differences between the modeled fits, we performed one-way ANOVA comparing the model output parameters assigning significant differences when the p value was < 0.05.

Figure 1: Absorbed peak Photosynthetically Usable Radiation (PUR) (µmol photons m^-2 s^-1) vs. peak Photosynthetically Active Radiation (PAR) (µmol photons m^-2 s^-1). The correlation between PAR, plotted on the x-axis and PUR, plotted on the y-axis, are coloured for each growth spectral waveband; 450 nm (blue circles), 660 nm (red circles) and white LED (black circles). The grey dashed line represents a hypothetical one to one correlation. A. is Prochlorococcus marinus MED4. B. is Prochlorococcus marinus SS120. C. is Prochlorococcus marinus MIT9313.

Prochlorococcus comparative genomics

We filtered the dataset of Omar et al. [53], for Enzyme Commission Numbers (EC numbers), or Kegg Orthology Numbers (KO numbers) identified by BRENDA [37] as ‘natural substrates’ for O₂; EC numbers identified by BRENDA as being activated, or inhibited by light; and EC numbers annotated by BioCyc [54] as corresponding to the Gene Ontology Term (GO:0006281 - DNA repair), in P. marinus strains (MED4, MIT9313, SS120, and NATL2A). We grouped orthologs together by EC number and their KO number and determined the occurrences of individual orthologs encoding each EC number, or KO number when EC number was not available, in a given strain. We merged the dataset with a list of enzyme Michaelis constant (K_m) values from other organisms, as K_m values from Prochlorococcus were only available in the case of Ribulose bisphosphate carboxylase. Gene counts for Flavodiirons were obtained from Allahverdiyeva et al. [55], as they do not have allocated EC numbers. A full list of enzymes and corresponding EC and KO numbers can be found in Table 2.

Results and discussion

Detection of Prochlorococcus proteins across O₂ and light niches in the ocean

Proteins from 13 annotated strains of P. marinus were detected across depths and oxygen concentrations in the ocean proteins data set analyzed. We focused our analysis here on photosynthetic protein complexes, grouped by clade. Figure 2 plots the observation of core photosynthetic complexes (Photosystem II, the Cytochromeb6f complex, Phototsystem I, ATP Synthase and Rubisco) across clades, as a function of depth (a proxy for light intensity) and measured [O₂]. Photosynthetic complexes from HLI (ex. MED4) were detected throughout the water column, predominantly at high [O₂]. Complexes from clade LLII/III (ex. SS120) and clade LLIV (ex. MIT9313) were detected throughout the water column in high [O₂] and at depth in low [O₂] samples. The exclusive assignment of ATP Synthase peptides to clade LLIV (ex. MIT9313) may result from the high conservation of protein sequence for this complex.

XXX More Text XXX from Amanda & Aurora XXX Proteins derived from clade HLXXX ecotypes of P. marinus were detected in OMZ at depths up to 200 meters, with O₂ of 15 µM. The extent to which [O₂] defines the niches occupied by different P. marinus ecotypes, as compared to potentially covarying environmental variables like photoperiod, light spectrum, and light level, is poorly described.

Figure 2: Ocean detection of Prochlorococcus marinus photosynthesis complexes. Protein detections (annotated as solid grey circles) are plotted vs. O₂ (µM) (X axis) and depth (m) (Y axis) at sample origin. Rows separate data annotated as from Prochlorococcus clades: HLI (P. marinus MED4 protein detection annotated as solid black circles), LLI (P. marinus NATL2A protein detection annotated as solid black circles), LLII/III (P. marinus SS120 protein detection annotated as solid black circles) and LLIV (P. marinus MIT9313 protein detection annotated as solid black circles). Columns show detections of proteins annotated as Photosystem II (PSII), Cytochromeb6f complex (Cytb6f), Photosystem I (PSI), ATP Synthase or Ribulose-1,5-bisphosphate oxygenase carboxylase (RUBISCO). For comparison culture growth experimental conditions are indicated by horizontal grey lines for depths approximating imposed peak Photosynthetically Active Radiation (µmol photons m^-2 s^-1); and vertical grey lines for [O₂] (µM). Data obtained from OceanProteinPortal (https://www.oceanproteinportal.org/).

Prochlorococcus marinus growth responses to photoperiod, PAR, spectral band, and [O₂]

Guided by the evidence of ocean distributions of proteins from Prochlorococcus we set up a matrix of photoperiods, PAR, spectral bands, and [O₂] to approximate current, and potential future, latitudinal, depth and seasonal niches for Prochlorococcus strains. As mentioned, growth under red light could prove mechanistically informative [51] to factors limiting Prochlorococcus growth, we therefore included the red spectral waveband even though it is not representative of Prochlorococcus niches. Although Prochlorococcus is currently limited to a narrow range of surface photoperiods, potential poleward latitudinal expansions, in combination with attenuation of light with depth, mean Prochlorococcus may potentially encounter a wide range of photoperiods. Our growth rate determinations generally agree with those from Moore et al. [56], for white LED and 250 µM O₂, but our study is, to our knowledge, the first to analyze the interactive growth responses of Prochlorococcus strains to varying [O₂], spectral wavebands and photoperiods.

Prochlorococcus marinus MED4, clade HLI, growth under 250 µM O₂ increased with higher imposed PAR and longer photoperiods (Figure 3), across all spectral wavebands. No growth was observed under any imposed conditions under a 4 h photoperiod. The maximum growth rate (µ_max) was 0.68 d^-1 achieved under 180 µE blue light and 16 h photoperiod.

Similar to growth trends under 250 µM O₂, MED4 maintained at 25 µM O₂ showed fastest growth when the photoperiod was 16 h for each spectral waveband, across PAR levels (Figure 3). The µ_max was 0.65 d^-1 (Table 3) achieved under 180 µmol photons m^-2 s^-1 blue light and 16 h photoperiod. The 4 h photoperiod experiments under white LED light were not performed as no growth was achieved when grown under an 8 h photoperiod of white LED light.

MED4 did not grow when sparged to the lowest [O₂] of 2.5 µM (Figure 3). 2.5 µM O₂ growth experiments were not conducted for 4 and 16 h photoperiods, as no reproducible growth occurred when MED4 was exposed to 8 and 12 h photoperiods.

Figure 3: chlorophyll specific growth rate (d^-1) for Prochlorococcus marinus MED4 (High-Light (HLI) near surface clade) vs. photoperiod (h). Rows show levels of imposed dissolved O₂ concentrations as 250 µM, 25 µM and 2.5 µM. Columns show 3 levels of growth Photosynthetically Active Radiation (PAR); 30, 90 and 180 µmol photons m^-2 s^-1 colours represent the actinic spectral waveband (nm). Large circles show mean or single determinations of growth rate from logistic curve fits; small circles show values for replicate determinations, if any: replicates often fall with larger circles.

The GAM model in Figure 4 summarizes MED4 growth responses to red (A,B) or blue (C,D) peak PAR and photoperiod across 2 imposed oxygen concentrations. Under 250 µM O₂ MED4 achieved fastest growth rates above peak blue light of ~180 µmol photons m^-2 s^-1, and the longest photoperiod of 16, indicated by the 0.64 d^-1 contour line representing the 90^th percentile of maximum achieved growth rate (Figure 4C). Growth decreased with decreasing photoperiod and decreasing peak PAR. Under red light growth was generally slower but the pattern of growth responses to photoperiod and PAR was similar (Figure 4A). Note the exclusion of MED4 from growth under 4 h photoperiod under both red and blue light (Figure 4). Under 25 µM O₂ MED4 showed similar growth responses, but was excluded from both 4 and 8 h photoperiods. MED4 did not grow under 2.5 µM O₂, so no GAM model was run. Considering the range of PAR levels, and spectral bands that MED4 can utilize, MED4 can inhabit not just shallow depths, where light levels are high, but also deeper regions, characterized by a lower level of blue light, subject to the limitation of a photoperiod of more than 4 h, even after depth attenuation of light. The photoregimes of winter temperate zones, due to shorter photoperiods, exclude MED4 from growth at any depth, however temperate photoperiods and light levels for the remainder of the year are potentially adequate to support MED4 growth, if water temperatures warm into the clade HLI tolerance range.

Figure 4: A contour plot of a Generalized Additive Model (GAM) representing the chlorophyll specific growth rate (d^-1) for Prochlorococcus marinus MED4 grown under 660 nm (red) or 450 nm (blue) light. X-axis is photoperiod (h). Y-axis is actinic Photosynethetically Active Radiation (PAR, µmol photons m^-2 s^-1). A. represents the model under 250 µM of O₂ and red light. B. represents the model under 25 µM of O₂ and red light. C. represents the model under 250 µM of O₂ and blue light. D. represents the model under 25 µM of O₂ and blue light. Legends represent a colour gradient of growth rate from no growth (white) to 1.00 d^-1 (dark red or dark blue). Labeled contour lines indicate the 90%, 50%, and 10% quantiles for achieved growth rate.

Prochlorococcus marinus SS120 clade LLII/III, growth under 250 µM O₂ increased with longer photoperiods, under 30 µmol photons m^-2 s^-1 peak PAR and across all spectral wavebands (Figure 5). No growth was observed under any blue light photoperiods when exposed to peak PAR of 90 µmol photons m^-2 s^-1 or greater. Growth rate, however increased with increasing photoperiods for white and red light under peak PAR of 90 µmol photons m^-2 s^-1 but showed growth inhibition at 16 h red light photoperiod. Growth rate decreased with longer photoperiods and showed growth inhibition at 12 and 16 h photoperiods under PAR of 180 µmol photons m^-2 s^-1 white LED, red or blue light. The µ_max was 0.5 d^-1 (Table 3) achieved under 90 µmol photons m^-2 s^-1 white LED light and 16 h photoperiod.

Under 25 µM O₂ and PAR of 30 µmol photons m^-2 s^-1 growth trends were similar to 250 µM O₂. SS120 showed no growth under a 4 h photoperiod for red spectral waveband, however under blue light, SS120 was able to grow (Figure 5). In contrast to the growth trends of the 250 µM O₂ and PAR of 90 µmol photons m^-2 s^-1 experiments, SS120 grew under 4 and 8 h blue light and 16 h red light photoperiods, however the growth rate decreased under 12 and 16 h white LED light photoperiod treatments. Blue light treatments under PAR of 180 µmol photons m^-2 s^-1 showed growth only under an 8 h photoperiod. The µ_max was 0.45 d^-1 (Table 3) achieved under 90 µmol photons m^-2 s^-1 blue light and 8 h photoperiod. The 25 µM O₂, less than 16 h photoperiod and 180 µmol photons m^-2 s^-1 under white LED light experiments were not performed due to time constraints.

SS120 did not reproducibly grow when sparged to the lowest O₂ of 2.5 µM (Figure 5). 2.5 µM O₂ growth experiments were not conducted for 4 and 16 h photoperiods under PAR of 180 µmol photons m^-2 s^-1, as no growth occurred when SS120 was exposed to 8 and 12 h photoperiods. Red light 16 h photoperiod experiments were not performed due to time constraints.

Figure 5: chlorophyll specific growth rate (d^-1) for Prochlorococcus marinus SS120 (Low-Light (LLII/III) deep ocean clade) vs. photoperiod (h). Rows show levels of imposed dissolved O₂ concentrations as 250 µM, 25 µM and 2.5 µM. Columns show 3 levels of growth Photosynthetically Active Radiation (PAR); 30, 90 and 180 µmol photons m^-2 s^-1 colours represent the actinic spectral waveband (nm). Large circles show mean or single determinations of growth rate from logistic curve fits; small circles show values for replicate determinations, if any: replicates often fall with larger circles.

The GAM model in Figure 6 summarizes growth responses of SS120 to red (A,B) or blue (C,D) peak PAR and photoperiod, across the 2 imposed oxygen concentrations. Under 250 µM O₂, Figure 6C showed highest growth rates below blue light PAR of 50 µmol photons m^-2 s^-1 and photoperiods between 8 and 12 h, indicated by the contour line labeled 0.19 d^-1 (representing the 90^th percentile of achieved growth rate). Under 250 µM O₂ SS120 is constrained to deeper ocean waters through its intolerance of higher blue PAR levels. These findings align with Moore et al. [56] and are expected for a low light clade. The disjunct regions of the GAM plot results from variable growth success of SS120 under 250 µM O₂. Growth rate patterns under red light were similar, although somewhat faster. In contrast, under 25 µM O₂ and a photoperiod of 8 h SS120 exploited all blue peak PAR levels, achieving faster growth rates at a higher PAR of ~100 µmol photons m^-2 s^-1, indicated by the contour line labeled 0.4 d^-1 (representing the 90^th percentile of achieved growth rate), out pacing the 90^th percentile fastest growth rates under 250 µM O₂ (Figure 6D). Under red light and 25 µM O₂ (Figure 6B) SS120 grew across most conditions of peak PAR and photoperiod, achieving fastest growth under long photoperiods and peak PAR between 50 ~100 µmol photons m^-2 s^-1. Thus, the designation of SS120 as a LL strain is dependent upon the [O₂]. SS120 did not, however, grow reliably under tested conditions at 2.5 µM O₂.

Figure 6: Contour plot of a Generalized Additive Model (GAM) representing the chlorophyll specific growth rate (d^-1) for Prochlorococcus marinus SS120 grown under 660 nm (red) or 450 nm (blue) light. X-axis is photoperiod (h). Y-axis is actinic Photosynethetically Active Radiation (PAR, µmol photons m^-2 s^-1). A. represents the model under 250 µM of O₂ and red light. B. represents the model under 25 µM of O₂ and red light. C. represents the model under 250 µM of O₂ and blue light. D. represents the model under 25 µM of O₂ and blue light. Legends represent a colour gradient of growth rate from no growth (white) to 1.00 d^-1 (dark red or dark blue). Labeled contour lines indicate the 90%, 50%, and 10% quantiles for achieved growth rate.

Prochlorococcus marinus MIT9313, clade LLIV, growth under 250 µM O₂ increased with longer photoperiods, under low 30 µmol photons m^-2 s^-1 peak PAR, (Figure 7). Under intermediate 90 µmol photons m^-2 s^-1 peak PAR growth rates decreased with increasing blue light photoperiods. Blue light did not induce growth at 180 µmol photons m^-2 s^-1 peak PAR, while MIT9313 showed only marginal growth under white LED and red light at 180 µmol photons m^-2 s^-1 peak PAR, under the 8 h photoperiod, consistent with Moore et al. [31]. The µ_max was 0.54 d^-1 achieved under 30 µmol photons m^-2 s^-1 blue light and 16 h photoperiod.

For MIT9313 under 25 µM O₂, growth rate increased with increasing photoperiods for all spectral wavebands tested (Figure 7), with the fastest overall growth rate for MIT9313 1.01 d^-1 achieved under peak PAR of 90 µmol photons m^-2 s^-1 and 16 h white LED light photoperiod. In marked contrast to the 250 µM O₂ growth experiments, MIT9313 grew when exposed to peak PAR of 180 µmol photons m^-2 s^-1 and blue light under all photoperiods except 16 h; additionally, white LED and red light treatments induced growth across all tested photoperiods under 25 µM O₂. The 25 µM O₂, 4 h photoperiod experiments under white LED light and were not performed due to time constraints.

MIT9313 grew under 2.5 µM O₂ particularly under blue LED light, albeit generally slower than under the parallel experiments at 25 µM O₂ (Figure 7). Growth estimates showed scatter among replicates, suggesting 2.5 µM O₂ is near the tolerance limit for growth of MIT9313. Growth rates increased with longer photoperiods under blue light treatments and peak PAR of 90 µmol photons m^-2 s^-1 but did not grow under 16 h photoperiod. Growth for MIT9313 under PAR of 180 µmol photons m^-2 s^-1 and blue light treatment decreased with increasing photoperiods with full growth inhibition under a 16 h photoperiod. The red light peak PAR of 180 µmol photons m^-2 s^-1 showed similar growth rates to blue light for 8 and 12 h photoperiods. The µ_max was 0.45 d^-1 achieved under 12 h blue light photoperiod and PAR of 90 µmol photons m^-2 s^-1. The 2.5 µM O₂ white LED treatments under 4, 8 and 16 h photoperiods and red light under 4 and 16 h photoperiods were not performed due to time constraints.

Figure 7: chlorophyll specific growth rate (d^-1) for Prochlorococcus marinus MIT9313 (Low-Light (LLIV) deep ocean clade) vs. photoperiod (h). Rows show levels of imposed dissolved O₂ concentrations as 250 µM, 25 µM and 2.5 µM. Columns show 3 levels of growth Photosynthetically Active Radiation (PAR); 30, 90 and 180 µmol photons m^-2 s^-1 colours represent the actinic spectral waveband (nm). Large circles show mean or single determinations of growth rate from logistic curve fits; small circles show values for replicate determinations, if any: replicates often fall with larger circles.

The GAM model in Figure 8 summarizes MIT9313 growth responses to red (A,B,C) or blue (D,E,F) peak PAR and photoperiod. Under 250 µM O₂, Figure 8D shows MIT9313 achieves fastest growth rates between blue peak PAR of 30 µmol photons m^-2 s^-1 and 50 µmol photons m^-2 s^-1 and photoperiods longer than 8 h, indicated by the contour line labeled 0.52 d^-1 representing the 90^th percentile of achieved growth rates. Figure 8D also shows that growth rate increases with longer photoperiods, as long as the blue peak PAR levels remain below 50 µmol photons m^-2 s^-1. In contrast, under red light and 250 µM O₂ MIT9313 grows faster while exploiting higher peak PAR and longer photoperiods. Figure 8E shows that MIT9313can exploit all blue PAR levels and most photoperiods with 90^th percentile of fastest growth rate between 30 to 100 µmol photons m^-2 s^-1 PAR. Figure 8F shows that MIT9313 maintains growth even under 2.5 µM O₂, under photoperiods between 4 and 8 h and peak blue PAR between 50 to 100 µmol photons m^-2 s^-1 PAR. Thus the designation of MIT9313 as a LL clade is dependent upon [O₂] and light spectra. (Figure 8E).

Figure 8: Contour plot of a Generalized Additive Model (GAM) representing the chlorophyll specific growth rate (d^-1) for Prochlorococcus marinus MIT9313 grown under 660 nm (red) or 450 nm (blue) light. X-axis is photoperiod (h). Y-axis is actinic Photosynthetically Active Radiation (PAR, µmol photons m^-2 s^-1). A. represents the model under 250 µM of O₂ and red light. B. represents the model under 25 µM of O₂ and red light. C. represents the model under 2.5 µM of O₂ and red light. D. represents the model under 250 µM of O₂ and blue light. E. represents the model under 25 µM of O₂ and blue light. F. represents the model under 2.5 µM of O₂ and blue light. Legends represent a colour gradient of growth rate from no growth (white) to 1.00 d^-1 (dark red or dark blue). Labeled contour lines indicate the 90%, 50%, and 10% quantiles for achieved growth rate.

PUR and growth responses

Cumulative diel PUR can potentially collapse photoperiod, PAR and spectral wavebands to a common metric of photosynthetically active light absorbed per day. Cumulative diel PUR dose (µmol photons m^-2 d^-1) was calculated from the imposed PUR (µmol photons m^-2 s^-1) and photoperiod (h). We plotted growth rates vs. cumulative diel PUR to determine whether growth is a simple response to diel PUR, across imposed photoperiods and spectral wavebands, or whether spectral wavebands or photoperiods have specific or interactive influences on growth beyond cumulative diel PUR.

Due to the absorption of P. marinus pigments in the blue spectral waveband range, the maximum cumulative diel PUR under blue light is almost 3 times that of white LED light, and about 5 times the red light treatment (Figure 1), despite being derived from the same photoperiods and peak PAR regimes. As such, only blue light experiments extend beyond cumulative diel PUR of ~ 2 x 10⁶ µmol photons m^-2 d^-1. This bias in the range of data leads us to caution in comparing model fits of growth in response to cumulative diel PUR under red vs. blue wavebands.

MED4 clade HLI

The representative of HLI clade, P. marinus MED4, showed no growth under any 4 h photoperiod treatments, even when a 4 h photoperiod delivered cumulative diel PUR equivalent to other photoperiod treatments (Figure 16A,B,C). In parallel MED4 showed no growth under 2.5 µM O₂, no matter the level of diel cumulative PUR. In contrast, under 250 or 25 µM O₂, and under any photoperiod greater than 4 h, MED4 growth under blue light was well described by a saturating response of growth [45] to increasing cumulative diel PUR, with saturation of growth rate achieved around 1.0 x 10⁶ µmol m^-2d^-1 (Figure 9A,B), and no evidence of inhibition of growth at any achieved cumulative diel PUR. Under the ‘artificial’ growth treatment of red light, MED4 achieved more growth per unit diel cumulative PUR (Figure 9A,B), consistent with Murphy et al. [51], who showed a lower cost for growth under red light, for MED4, because red light provokes less photoinactivation of PSII, than equivalent levels of blue light. For distinct fits for different photoperiods refer to Figure 16 A,B and C.

SS120 clade LLII/III

The representative of the LLII/III clade, P. marinus SS120 showed almost no growth under 2.5 µM O₂ experiments (Figure 16F). Most 4 h photoperiod treatments of SS120 also did not grow under 250 µM O₂, even when a 4 h photoperiod delivered cumulative diel PUR equivalent to other photoperiod treatments (Figure 16D). SS120 did not grow when exposed to more than ~1.0 x 10⁶ µmol photons m^-2 d^-1 of cumulative diel PUR under any spectral waveband or photoperiod combination, under 250 µM O₂ experiments (Figure 16D).

Under both 25 and 250 µM O₂ experiments, SS120 growth plateaued by about 5.0 x 10⁵ µmol photons m^-2 d^-1 diel PUR, with some scatter among photoperiod and spectral waveband regimes. The onset of growth inhibition extended to higher cumulative diel PUR for cultures under 25 µM O₂, showing that SS120 is partially protected from photoinhibition of growth by 25 µM O₂. Under 25 µM O₂, red light again generated more growth of SS120 per unit cumulative diel PUR, than did blue light, again consistent with lower cost of growth through lower photoinactivation under red light (Figure 9E). For distinct fits for different photoperiods refer to Figure 16 D,E and F.

MIT9313 clade LLIV

The LLIV clade representative, P. marinus MIT9313, showed growth rising to a plateau by about 5 x 10⁵ µmol photons m^-2 d^-1 of cumulative diel PUR, with higher growth rates over a narrower plateau under 25 and 250 µM O₂ (Figure 16H,G), compared to a wider, lower, flatter response to cumulative diel PUR under 2.5 µM O₂ (Figure 16I). Above about 1.0 x 10⁶ µmol photons m^-2 d^-1 of cumulative PUR under 250 µM O₂, MIT9313 showed full inhibition of growth, across photoperiods, and spectral wavebands (Figure 16G). In contrast, under 25 µM O₂, MIT9313 showed a greatly extended exploitation of higher cumulative diel PUR, with full growth inhibition only above about 3.5 x 10⁶ µmol photons m^-2 d^-1 (Figure 16H). Similarly, under 2.5 µM O₂, MIT9313 grew more slowly, but only showed full growth inhibition above about 3.5 x 10⁶ µmol photons m^-2 d^-1 cumulative diel PUR (Figure 16I).

As with MED4 and SS120, our data again support enhanced growth under conditions of low cumulative diel PUR and 660 nm (red) spectral bandwidth, consistent with Murphy et al.[51] who found a lower cost of growth, due to decreased photoinactivation of PSII under red, compared to blue, wavebands (Figure 9G,H). Interestingly, this protective effect of red light disappears for MIT9313 growing under 2.5 µM O₂, possibly because photoinactivation is strongly suppressed under this low [O₂] (Figure 9I). For distinct fits for different photoperiod fits refer to Figure 16 G,H and I.

Figure 9: chlorophyll specific growth rate (d^-1) vs. cumulative diel Photosynthetic Usable Radiation (PUR, µmol photons m^-2 d^-1). Rows show levels of imposed dissolved O₂ concentrations as 250 µM, 25 µM and 2.5 µM. Columns are strains; MED4 (A,B,C), SS120 (D,E,F) and MIT9313 (G,H,I). Shapes show the imposed photoperiod (h); 4 h (solid square), 8 h (solid diamond), 12 h (solid circle), 16 h (solid upright triangle). Symbol colours show the spectral waveband for growth; 660 nm (red symbols), and 450 nm (blue symbols). Large symbols show mean of growth rate from logistic curve fits; small symbols show values for replicate determinations, if any. Harrison and Platt [45] 4 parameter model fit to 660 nm (red lines) and 450 nm (blue lines) growth data for each combination of strain and dissolved oxygen shown with solid lines (significantly different fits, p value < 0.05) or dashed lines (not significantly different fits, p value > 0.05) using one-way ANOVA.

Photosystem II maintenance, oxygen metabolism, and DNA repair as limitations on Prochlorococcus growth

Prochlorococcus remain challenging to culture, as their reduced genomes – the smallest of any known oxyphototroph – render them partially dependent upon mutualistic heterotrophic bacteria to detoxify reactive oxygen species [57,58]. MED4, SS120 and MIT9313 have been successfully cultured in laboratories [43,56], and used to show that ecotypic classifications correspond to biochemical differences among strains [42].

Under full atmospheric [O₂] and blue light, LL clades of Prochlorococcus are restricted to growth under low light, in part because they suffer photoinhibition of Photosystem II (PSII) through several paths, including direct absorbance of UV or blue light, in parallel with generation of Reactive Oxygen Species (ROS) if the electron flow is slowed [59], producing damaging singlet oxygen (¹O₂) [51,59–61]. Repair of photoinactivated PSII relies on the removal of damaged PsbA [62,63], followed by reassembly with newly synthesized PsbA [64]. Degradation of PsbA is a rate-limiting step in recovery from photoinhibition [65], mediated largely by a heterohexamer of (FtsH1-FtsH2)₃, a membrane-bound [66,67] metalloprotease [(Chiba et al., 2002) (Yoshioka-Nishimura and Yamamoto, 2014), 68,69], XXX (AMANDA REF from ALGATECH) XXX.

Prochlorococcus genomes encode 4 FtsH proteins, henceforth referred to as FtsH1-4, homologs to the characterized FtsH isoforms of the model freshwater cyanobacterium Synechocystis FtsH, and with presumably parallel functions (Table 1). Upon a shift to higher light HLI MED4 upregulated expression of FtsH1 and FtsH2 [42], homologs to the Synechocystis slr0228 and slr1604, implicated in PSII repair. In contrast, representative LLIV strain MIT9313 showed less overall expression of the FtsH proteases, and thus has fewer FtsH serving each photosystem. Furthermore, MIT9313 expressed primarily FtsH3, homologous to Synechocystis slr1463, possibly involved in PSI biogenesis, and FtsH expression did not increase in response to light stress in MIT9313 [42]. Through adaptation to steady low light, clade LLIV Prochlorococcus instead allocate resources to processes other than dynamic regulation of PSII repair.

Table 1: FtsH protease homologs in Prochlorococcus marinus and the model cyanobacterium Synechocystis sp. PCC6803. Protein homology determined by multiple sequence alignment with MUSCLE followed by construction of maximum likelihood phylogenetic tree using 1000 bootstrap replicates in MEGAX.

Organism	Homolog 1	Homolog 2	Homolog 3	Homolog 4
Prochlorococcus marinus	FtsH1	FtsH2	FtsH3	FtsH4
Synechocystis sp. PCC6803	Slr0228	Slr1604	Sll1463	Slr1390
Function	PSII Repair	PSII Repair	PSI biogenesis?	Cell viability

Ocean detections of proteins mediating protein metabolism support this interpretation of distinct FtsH function across clades of P. marinus. Ribosome proteins from clade HLI MED4, clade LLI NATL2A, clade LLII/III SS120 and clade LLIV MIT9313 show generally similar patterns vs. [O₂] and depth, a proxy for peak PAR (Figure 10). FtsH3, inferred to mediate PSI assembly, likewise shows a similar pattern between MED4 and MIT9313 (Figure 10). But only MED4 shows detected presence of the FtsH1 & FtsH2 isoforms inferred to mediate PSII repair, and then only in near-surface samples subject to higher light. Furthermore, even though MIT9313 grows (Figure 8), and is detected in the ocean at low [O₂] (Figure 2), no FtsH from MIT9313 is detected at low [O₂] (Figure 10), suggesting limited requirement for protein turnover under low [O₂].

Figure 10: Ocean detection of Prochlorococcus marinus protein metabolism complexes. Protein detections (annotated as solid grey circles) are plotted vs. O₂ (µM) (X axis) and depth (m) (Y axis) at sample origin. Rows separate data annotated as from Prochlorococcus clades: HLI (P. marinus MED4 protein detection annotated as solid black circles), LLI (P. marinus NATL2A protein detection annotated as solid black circles), LLII/III (P. marinus SS120 protein detection annotated as solid black circles) and LLIV (P. marinus MIT9313 protein detection annotated as solid black circles). Columns show detections of proteins annotated as FtsH Protease Complexes (FtsH1, FtsH2, FtsH3) or the Ribosome. Culture growth experimental conditions indicated by horizontal grey lines for depths approximating Photosynthetically Active Radiation (µmol photons m^-2 s^-1) and vertical grey lines for [O₂] (µM). Data obtained from OceanProteinPortal (https://www.oceanproteinportal.org/).

Figure 11 shows the measured or inferred K_M for [O₂] for genes encoding enzymes [53] for P. marinus strains from clades HLI, LLI, LLII/III and LLIV. MED4 increases expression of alternative oxidase (‘ubiquinol oxidase (non electrogenic)’) to cope with changes in light [70], by dissipating electrons from the inter-system transport chain. The approximate K_M for [O₂] of ~ 25 µM for ubiquinol oxidase (non electrogenic) (Figure 11) is comparable to the lower limit for growth of MED4 in our experiments (Figure 4). We suggest that dependence upon this enzyme excludes MED4 from low oxygen zones. The genome scan shows SS120 and MIT9313 lack this gene (Figure 11), and therefore, lack this oxygen-dependent path to cope with changing excitation. Conversely, a gene encoding (S)-2-hydroxy-acid oxidase is encoded in the MIT9313 genome (Figure 11). (S)-2-hydroxy-acid oxidase catalyzes the reaction of 2-hydroxy acid with O₂ to produce toxic H₂O₂ [71]. (S)-2-hydroxy-acid oxidase has an approximate K_M for [O₂] of ~ 250 µM, and produces H₂O₂, so growth at lower [O₂] may protect MIT9313 from auto-intoxication from production of H₂O₂. We hypothesize that under 250 µM O₂ and higher blue light, P. marinus MIT9313 suffered photoinhibition, resulting from the inactivation of PSII caused by the production of the reactive oxygen species, hydrogen peroxide. This photoinhibition is compounded by the limited inducible repair mechanism for PSII, due to the absence of FtsH 1 and 2 expression in P. marinus MIT9313 [42]. We hypothesize that under the conditions of our high light and 2.5 µM or 25 µM O₂ experiments, the activity of the (S)-2-hydroxy-acid oxidase enzyme is suppressed. As a result, the catalyzed production of hydrogen peroxide is inhibited, leading to less PSII damage, allowing MIT9313 to avoid photoinhibition and circumvent its limitations on PSII repair to exploit higher light. Figure 11 also shows that P. marinus SS120 is the only tested ecotype to lack the pyridoxal 5’-phosphate synthase enzyme. The pyridoxal 5’-phosphate synthase enzyme is an important cofactor in the biosynthesis of vitamin B₆ [72]. Vitamin B₆ is a potential antioxidant and can effectively quench singlet oxygen [73]. The absence of the pyridoxal 5’-phosphate synthase enzyme may explain why P. marinus SS120 does not grow as well as P. marinus MIT9313, when exposed to high light stress under 25 µM O₂ and not at all under 2.5 µM O₂ (Figure 5).

Figure 11: K_m values for oxygen metabolizing enzymes. The y-axis represents the log10 concentration of oxygen substrate (µM). The x-axis represents the oxygen metabolizing enzymes encoded in at least one of the Prochlorococcus marinus strains in this study. The Prochlorococcus marinus strains are indicated in rows. The solid circles represent K_m values from literature and the asterisks represent predicted values. colours represent the gene counts. The red shaded area denotes a K_m oxygen concentration range from 230 to 280 µM. The green shaded area denotes a K_m oxygen concentration range from 5 to 50 µM . The blue shaded area denotes a K_m oxygen concentration range from 0.5 to 5 µM. The black bars show the minimum and maximum K_m values. Figure was generated using a filtered subset of the annotated phytoplankton gene sequences dataset from Omar et al. [53].

Figure 12 shows genes encoding DNA repair for P. marinus strains. As expected, P. marinus MED4 possesses the largest, most complete suite of genes encoding DNA repair enzymes, followed by P. marinus MIT9313. Conversely, P. marinus SS120 demonstrates the smallest genomic capacity for DNA repair. Prochlorococcus marinus MED4 and NATL2A were the only strains to possess a gene encoding deoxyribodipyrimidine photolyase, which, in the presence of blue light, is responsible for repairing DNA damaged by UV light [74]. Prochlorococcus marinus MED4 was also the only strain to possesses a gene encoding DNA ligase, which uses ATP as a cofactor for DNA repair. The absence of genes encoding deoxyribodipyrimidine photolyase and DNA ligase (ATP) in P. marinus MIT9313 and P. marinus SS120 explain why these two strains cannot tolerate growth under full [O₂] and high light, found at the ocean surface. Furthermore, the protective effect of lower [O₂], allowing these strains to grow at higher light, may relate in part to suppression of DNA damage when generation of Reactive Oxygen Species is suppressed at lower [O₂]. NATL2A, a clade LLI, has been found near the ocean surface during deep ocean mixing [75]. Malmstrom et al. [75] attributes NATL2A tolerance to short exposures of high light to the presence of the genes encoding photolyase, a gene found in HL clades. The presence of deoxyribodipyrimidine photolyase and absence of DNA ligase (ATP) supports why NATL2A tolerates limited exposure to high light and why NATL2A is unable to fully repair damaged DNA. Prochlorococcus are highly susceptible to hydrogen peroxide (H₂O₂) toxicity as they lack genes which scavenge H₂O₂ molecules [57]. The small cell size of Prochlorococcus allow the reactive oxygen species (ROS), H₂O₂, to cross the cell membrane [76]; however, accumulation of extracellular H₂O₂ remains toxic to Prochlorococcus [57,58].

Figure 12: Genes encoding DNA repair enzymes. The y-axis represents Prochlorococcus marinus strains. The x-axis represents enzymes encoded for DNA repair found in at least one Prochlorococcus marinus strain in this study. Point size indicate gene counts. Figure was generated using a filtered subset of the annotated phytoplankton gene sequences dataset from Omar et al. [53].

The potential for niche expansion into temperate regions by P. marinus varies depending on the season, which influences achieved underwater photoperiods and light levels. Temperate summer delivers 11 hours of blue waveband light underwater, above the photic threshold of 20 µmol photons m^-2 s^-1, while temperate spring/fall delivers 8 hours of blue waveband light underwater, photoperiod ranges which are permissive for growth of all three P. marinus. In contrast temperate winter delivers only about 2 h of blue waveband light underwater above the photic threshold, which precludes growth of MED4 and SS120, even if winter waters reached permissive temperatures. MIT9313 and SS120 will be excluded from near-surface growth niches by high PAR, unless OMZ zones extend to the near surface.

Diverse P. marinus strains [5] differentially exploit potential photoregimes, both at the surface and deep in the water column. Some P. marinus strains grow under low oxygen environments, similar to OMZ. The LL clades we tested can function as ‘HL’ in oxygen environments of 25 µM, and as low as 2.5 µM, in the case of MIT9313.

West et al. [westNichePartitioningProchlorococcusPopulationsStratified1999?] and Malmstrom et al. [75] found that decreased abundances of the LL clades corresponded to increased depth of the surface mixed layer. Malmstrom et al. [75] attributes the transport of LL ecotypes to the surface and consequent exposure to photoinhibitory high light levels as the reason for low cell abundances with increased mixed layer depth. West et al. [westNichePartitioningProchlorococcusPopulationsStratified1999?] found the depth of the mixed layer strongly influenced the depth transition from HL to LL clades, but that factors other than light levels may influence the variations in the upper and lower depth limits of these ecotypes. We hypothesize that low cell abundances of LL ecotypes in the mixed layer is likely driven in part by increased [O₂], and it is [O₂] that constrains LL clades to deeper waters, not necessarily the light level. We found that under 25 µM O₂ representatives of ‘LL’ clades, SS120 and MIT9313, actually tolerate approximately 1.0 x 10⁶ µmol photons m^-2 d^-1 of PUR (Figure 16E,H), comparable to the representative HL clade, MED4 which also exhibited growth saturation at the same cumulative diel PUR of 1.0 x 10⁶ µmol photons m^-2 d^-1 (Figure 16A,B). Growth under lower O₂ allowed MIT9313 to substantially increase its exploitation of higher diel PUR (Figure 16I).

Summary and conclusions

We analyzed growth rates to determine the viability of P. marinus MED4, a clade HLI ecotype found near the ocean surface, P. marinus SS120, a clade LLII/III ecotype found deep in the water column, and P. marinus MIT9313, a clade LLIV ecotype also found in deep oceans including OMZ, under a matrix of spectral waveband, light level, photoperiod and oxygen conditions approximating present day and hypothetical future niches. Low light levels in the blue spectral waveband prevail in deep ocean waters [13,33], while solar incidence angle and light attenuation also progressively attenuate the photoperiod with depth beneath the ocean surface.

Prochlorococcus marinus MED4 has a physiological requirement for more than 4 h of light per day; thus this strain will not exploit habitats with short photoperiods, typical of temperate winter or light attenuated depths, even if water temperature warms into the clade HLI tolerance range. MED4 is also excluded from the lowest oxygen habitats, represented by our 2.5 µM O₂ experimental conditions. MED4 can, however, grow under OMZ regions with slightly higher [O₂], as demonstrated by the growth under our 25 µM O₂ experiments. Genomic analyses (Figure 11) and previous transcriptional analyses [70] suggest MED4 is excluded from growth below ~ 25 µM O₂ because it relies upon a ubiquinol oxidase, non-electrogenic, to maintain oxidation/reduction balance in the intersystem electron transport chain. On the other hand, MED4 shows inducible expression of FtsH isoforms [42], to counter photoinactivation of PSII under higher PAR and [O₂] environments. Photoinactivation, does, however, impose an increased cost of growth upon MED4, since growth under red light, to lower the rate of photoinactivation of PSII [51], allows MED4 to achieve faster growth per absorbed photon than growth under blue light. TARA Oceans Project data [19] indeed reported presence of P. marinus MED4-like genomes at depths ranging from 5 m to 90 m, representing high to low blue light levels, in the Pacific South East Ocean. Delmont and Eren [19] did not analyze data from depths beyond the subsurface chlorophyll maximum layer, the layer in the water column where the chlorophyll a concentration peaks, nor did they report the [O₂] at those depths. Our growth findings are also consistent with Figure 2 showing PSII proteins annotated as MED4, clade HLI, at depths up to 200 meters with O₂ of 15 µM.

Prochlorococcus marinus SS120, a LLII/III clade representative, showed an interactive inhibition of growth by oxygen and cumulative diel PUR, with a higher tolerance for higher cumulative diel PUR under 25 µM O₂, compared to 250 µM O₂. Thus, SS120 can exploit habitats with O₂ levels spanning 25 µM O₂ to 250 µM O₂, including higher PAR environments within OMZ. SS120 is likely excluded from the combination of higher [O₂] and higher PAR by genomic limitations on capacity for DNA repair (Figure 12), and possibly by limited capacity for synthesis of reactive oxygen quenchers (Figure 11). Our growth results are supported by Lavin et al. [10] who found evidence of LLII/III and LLIV ecotypes, using terminal restriction fragment length polymorphism analyses, at depths above 40 m, where light levels are higher, within OMZ, and by Figure 2 showing PSII protein subunits annotated as derived from SS120 at all depths ranging from 20 to 200 m and all [O₂] in an OMZ of the tropical North Pacific Ocean. SS120 grew under photoperiods longer than 4 h and showed increasing growth rate with increasing photoperiods. However, we found P. marinus SS120 can potentially exploit more diverse ecological niches within the ocean layers, even in regions with higher levels of blue spectral waveband, but only under O₂ of 25 µM (Figure 5). Lavin et al. [10] show evidence of LLII/III ecotypes at varying depths from 20 to 30 m and from 75 to 200 µM O₂ in the oxygen minimum zone (OMZ) of the tropical South Pacific Ocean, illustrating its tolerance of high light levels under low O₂. SS120 has the potential to thrive in deep temperate zones, specifically during the spring, summer, and fall seasons when the duration of daylight exceeds 4 h, if [O₂] are near surface saturation of about 250 µM. Under lower oxygen levels of 25 µM, SS120 can also potentially exploit a 4 h photoperiod in the blue waveband, and thus has the potential to inhabit a potential warmed, deep, temperate OMZ, during the winter season.

Prochlorococcus marinus MIT9313, a LLIV clade representative, shows potential to inhabit future warmer temperate zones year-round, as it grows under a 4 h photoperiod, expected in winter, or at light-attenuated depths. MIT9313 demonstrates an unexpected tolerance to higher light levels and cumulative diel PUR, but only under low oxygen conditions of 25 µM and 2.5 µM (Figure 7), enabling MIT9313 to grow in OMZ, even at depths closer to the surface. MIT9313 carries a gene encoding (S)-2-hydroxy-acid oxidase [71], with a K_M for [O₂] of ~ 250 µM (Figure 11), which produces H₂O₂. Growth at lower [O₂] may protect MIT9313 from auto-intoxication from production of H₂O₂. We hypothesize that under 250 µM O₂ and higher blue light, P. marinus MIT9313 suffers photoinhibition, resulting from the inactivation of PSII caused by the production of H₂O₂. This photoinhibition is compounded by the limited inducible repair for PSII, due to the absence of FtsH 1 and 2 expression in P. marinus MIT9313 [42]. MIT9313 shows remarkable ability to thrive under very low [O₂], potentially allowing it to expand into broader ecological niches. These results are supported by Figure 2 showing PSI protein subunits annotated as derived from MIT9313 detected at depths > 120 m, along with PSII subunits at depths from 50 m to 200 m in regions where O₂ was 15 µM. Bagby and Chisholm [77] suggest that O₂ has a protective role in Prochlorococcus under lower carbon dioxide environments when carbon fixation is limited. The deep water environments typical for MIT9313 are relatively nutrient rich, and Prochlorococcus take up and metabolize various sugars [78–80] and amino acids [81]. In future work we aim to test whether MIT9313 is using photosynthesis to drive CO₂ fixation in low O₂ environments, or whether PSII generation of O₂ acts as an electron sink for respiration, using ATP for maintenance and to take up nutrients from the surroundings. Partensky et al. [15] found that in the low-light conditions found in the OMZ, MED4, SS120 and MIT9313 all became net O₂ consumers, suggesting that the low light levels cause the respiratory chain to consume more O₂ than the photosynthetic electron transport chain generates, thus contributing to maintenance of the low O₂ environment.

Supplemental

Figure 13: PSI MCMIX-OD Multicultivator. Spectral waveband, light level and photoperiod are individually controlled for each culture tube. Real time Optical Density (OD) measurements eliminate intrusive subsampling of cultures. The temperature of culture tubes are collectively controlled via heating or cooling of the aquarium water. Gas with specific oxygen concentrations is bubbled through a humidifier and passed through a 0.2 um filter.

Figure 14: Fitting chlorophyll specific growth rate for each tube in the Multicultivator. The left y-axis is \(\Delta\)OD (OD680 - OD720). The right y-axis is actinic PAR levels (µmol photons m^-2 s^-1). The x-axis is time in hours (h). The green points are \(\Delta\)OD measurements taken every 5 minutes. The black lines are logistic growth curves fit using a nonlinear model regression (R package, minpack.lm). The gold points are the residuals of the fit. The blue or orange points represent the actinic spectral waveband under 450 nm or 660 nm, showing the imposed PAR level (µmol photons m^-2 s^-1) and photoperiod (h). Meta data associated with each Multicultivator tube are in columns.

Figure 15: Normalized absorbance and Photosynthetically Usable Radiation for Prochlorococcus marinus MED4 (A,D,G); SS120 (B,E,H); MIT9313 (C,F,I) grown under three emission wavebands. (A,B,C) Growth light emission spectra from the White LED (normalized to 439 nm; dotted black line); whole cell absorbance spectra (normalized to absorbance maxima between 400 nm and 460 nm; dashed purple line); and calculated PUR spectra (solid black line and shaded grey). (D,E,F) Growth light emission spectra at 660 nm (normalized to 647 nm; dotted red line); whole cell absorbance spectra (normalized to absorbance maxima between 400 nm and 460 nm; dashed purple line); and calculated PUR spectra (solid black line and shaded red). (G,H,I) Growth light emission spectra at 450 nm (normalized to 441 nm; dotted blue line); whole cell absorbance spectra (normalized to absorbance maxima between 400 nm and 460 nm; dashed purple line); and calculated PUR spectra (solid black line and shaded blue). Actinic PAR (µmol photons m^-2 s^-1) and calculated PUR (µmol photons m^-2 s^-1) levels are indicated.

Figure 16: Chlorophyll specific growth rate (d^-1) vs. cumulative diel Photosynthetic Usable Radiation (PUR, µmol photons m^-2 d^-1). Rows show levels of imposed dissolved O₂ concentrations as 250 µM, 25 µM and 2.5 µM. Columns are strains; MED4 (A,B,C), SS120 (D,E,F) and MIT9313 (G,H,I). Shapes show the imposed photoperiod (h); 4 h (solid square), 8 h (solid diamond), 12 h (solid circle), 16 h (solid upright triangle). Symbol colours show the spectral waveband for growth; white LED (black symbols), 660 nm (red symbols), and 450 nm (blue symbols). Large symbols show mean of growth rate from logistic curve fits; small symbols show values for replicate determinations, if any. Harrison and Platt [45] 4 parameter model fit to data pooled for each combination of strain and dissolved oxygen shown with solid lines. Separate models fit to photoperiod data and shown if significantly different (p value < 0.05) from the pooled model using one-way ANOVA; 4 h (long dashed line); 8 h (dotted line); 12 h (dashed line); and 16 h (dot dashed line).

Figure 17: Prochlorococcus genes encoding enzymes activated or inhibited by light. The y-axis represents Prochlorococcus marinus strains. The x-axis represents enzymes encoding light-dependent enzymes found in at least one Prochlorococcus marinus strain in this study. Point size indicate gene counts. Figure was generated using a filtered subset of the annotated phytoplankton gene sequences dataset from Omar et al. [53].

Table 2: Enzymes shown in Figures 11, 12 and 17 with their Enzyme Commission numbers (EC) and Kegg Orthology (KO).

Enzyme Name	EC	Kegg Orthology
quinate dehydrogenase	1.1.1.24	K09484
pyranose oxidase	1.1.3.10	K23272
L-sorbose oxidase	1.1.3.11	NA
pyridoxine 4-oxidase	1.1.3.12	K18607
alcohol oxidase	1.1.3.13	K17066
(S)-2-hydroxy-acid oxidase	1.1.3.15	K00104
(S)-2-hydroxy-acid oxidase	1.1.3.15	K11517
ecdysone oxidase	1.1.3.16	K10724
choline oxidase	1.1.3.17	K17755
secondary-alcohol oxidase	1.1.3.18	NA
4-hydroxymandelate oxidase (decarboxylating)	1.1.3.19	NA
long-chain-alcohol oxidase	1.1.3.20	K17756
long-chain-alcohol oxidase	1.1.3.20	NA
glycerol-3-phosphate oxidase	1.1.3.21	K00105
thiamine oxidase	1.1.3.23	NA
hydroxyphytanate oxidase	1.1.3.27	NA
nucleoside oxidase	1.1.3.28	NA
polyvinyl-alcohol oxidase	1.1.3.30	NA
D-arabinono-1,4-lactone oxidase	1.1.3.37	K00107
vanillyl-alcohol oxidase	1.1.3.38	K20153
nucleoside oxidase (H2O2-forming)	1.1.3.39	NA
glucose oxidase	1.1.3.4	NA
D-mannitol oxidase	1.1.3.40	NA
alditol oxidase	1.1.3.41	K00594
prosolanapyrone-II oxidase	1.1.3.42	K20550
aclacinomycin-N oxidase	1.1.3.45	K15949
4-hydroxymandelate oxidase	1.1.3.46	K16422
5-(hydroxymethyl)furfural oxidase	1.1.3.47	K16873
3-deoxy-alpha-D-manno-octulosonate 8-oxidase	1.1.3.48	K19714
hexose oxidase	1.1.3.5	K21840
cholesterol oxidase	1.1.3.6	K03333
aryl-alcohol oxidase	1.1.3.7	NA
L-gulonolactone oxidase	1.1.3.8	K00103
galactose oxidase	1.1.3.9	K04618
glycerol oxidase	1.1.3.B4	NA
(S)-2-hydroxyglutarate dehydrogenase	1.1.5.13	NA
decaprenylphospho-beta-D-ribofuranose 2-dehydrogenase	1.1.98.3	NA
cellobiose dehydrogenase (acceptor)	1.1.99.18	NA
glucooligosaccharide oxidase	1.1.99.B3	NA
catechol oxidase	1.10.3.1	K00422
ubiquinol oxidase (non-electrogenic)	1.10.3.11	K17893
grixazone synthase	1.10.3.15	K20204
superoxide oxidase	1.10.3.17	K12262
laccase	1.10.3.2	K00421
laccase	1.10.3.2	K05909
L-ascorbate oxidase	1.10.3.3	K00423
L-ascorbate oxidase	1.10.3.3	NA
o-aminophenol oxidase	1.10.3.4	K20204
o-aminophenol oxidase	1.10.3.4	K20219
3-hydroxyanthranilate oxidase	1.10.3.5	NA
rifamycin-B oxidase	1.10.3.6	NA
catechol 1,2-dioxygenase	1.13.11.1	K03381
7,8-dihydroxykynurenate 8,8a-dioxygenase	1.13.11.10	NA
tryptophan 2,3-dioxygenase	1.13.11.11	K00453
linoleate 13S-lipoxygenase	1.13.11.12	K00454
2,3-dihydroxybenzoate 3,4-dioxygenase	1.13.11.14	K10621
3,4-dihydroxyphenylacetate 2,3-dioxygenase	1.13.11.15	K00455
3-carboxyethylcatechol 2,3-dioxygenase	1.13.11.16	K05713
indole 2,3-dioxygenase	1.13.11.17	NA
persulfide dioxygenase;	1.13.11.18	K17725
cysteamine dioxygenase	1.13.11.19	K10712
catechol 2,3-dioxygenase	1.13.11.2	K00446
catechol 2,3-dioxygenase	1.13.11.2	K07104
4-hydroxyphenylpyruvate dioxygenase	1.13.11.27	K00457
protocatechuate 3,4-dioxygenase	1.13.11.3	K00448
protocatechuate 3,4-dioxygenase	1.13.11.3	K00449
arachidonate 15-lipoxygenase	1.13.11.33	K00460
arachidonate 15-lipoxygenase	1.13.11.33	K08022
arachidonate 15-lipoxygenase	1.13.11.33	K19246
arachidonate 5-lipoxygenase	1.13.11.34	K00461
acireductone dioxygenase (Ni2+-requiring)	1.13.11.53	K08967
linolenate 9R-lipoxygenase	1.13.11.61	K18031
all-trans-8’-apo-beta-carotenal 15,15’-oxygenase	1.13.11.75	K00464
7,8-dihydroneopterin oxygenase	1.13.11.81	K01633
peptide-aspartate beta-dioxygenase	1.14.11.16	K00476
taurine dioxygenase	1.14.11.17	K03119
procollagen-proline 4-dioxygenase	1.14.11.2	K00472
nitric oxide dioxygenase	1.14.12.17	K05916
salicylate 1-monooxygenase	1.14.13.1	K00480
cyclohexanone monooxygenase	1.14.13.22	K03379
violacein synthase	1.14.13.224	K20090
L-lysine N6-monooxygenase (NADPH)	1.14.13.59	K03897
magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase	1.14.13.81	K04035
kynurenine 3-monooxygenase	1.14.13.9	K00486
unspecific monooxygenase	1.14.14.1	K00490
dimethylsulfone monooxygenase	1.14.14.35	K17228
heme oxygenase (biliverdin-producing, ferredoxin)	1.14.15.20	K21480
choline monooxygenase	1.14.15.7	K00499
stearoyl-CoA 9-desaturase	1.14.19.1	K00507
acyl-lipid (n+3)-(Z)-desaturase (ferredoxin)	1.14.19.23	K10255
tetracycline 7-halogenase	1.14.19.49	K14257
tryptophan 7-halogenase	1.14.19.9	K14266
ferroxidase	1.16.3.1	NA
bacterial non-heme ferritin	1.16.3.2	NA
xanthine dehydrogenase	1.17.1.4	NA
(light-dependent) protochlorophyllide reductase	1.3.1.33	NA
coproporphyrinogen oxidase	1.3.3.3	NA
9,9’-dicis-zeta-carotene desaturase	1.3.5.6	NA
short-chain acyl-CoA dehydrogenase	1.3.8.1	NA
dihydroorotate dehydrogenase (fumarate)	1.3.98.1	NA
L-aspartate oxidase	1.4.3.16	NA
glycine oxidase	1.4.3.19	NA
D-amino-acid oxidase	1.4.3.3	NA
monoamine oxidase	1.4.3.4	NA
pyridoxal 5’-phosphate synthase	1.4.3.5	NA
nitrate reductase (NADH)	1.7.1.1	NA
ferredoxin-nitrate reductase	1.7.7.2	NA
cytochrome-c oxidase	1.9.3.1	NA
thymidylate synthase (FAD)	2.1.1.148	NA
5-aminolevulinate synthase	2.3.1.37	NA
aralkylamine N-acetyltransferase	2.3.1.87	NA
sucrose-phosphate synthase	2.4.1.14	NA
protein O-GlcNAc transferase	2.4.1.255	NA
15-cis-phytoene synthase	2.5.1.32	NA
4-(cytidine 5’-diphospho)-2-C-methyl-D-erythritol kinase	2.7.1.148	NA
crossover junction endodeoxyribonuclease	3.1.22.4	NA
3’,5’-cyclic-GMP phosphodiesterase	3.1.4.35	NA
phospholipase D	3.1.4.4	NA
DNA-3-methyladenine glycosylase II	3.2.2.21	NA
leucyl aminopeptidase	3.4.11.1	NA
glutamyl endopeptidase	3.4.21.19	NA
ribulose-bisphosphate carboxylase	4.1.1.39	NA
deoxyribodipyrimidine photo-lyase	4.1.99.3	NA
deoxyribodipyrimidine photo-lyase	4.1.99.3	NA
aldehyde oxygenase (deformylating)	4.1.99.5	NA
nitrile hydratase	4.2.1.84	NA
chorismate synthase	4.2.3.5	NA
DNA-(apurinic or apyrimidinic site) lyase	4.2.99.18	NA
lactoylglutathione lyase	4.4.1.5	NA
adenylate cyclase	4.6.1.1	NA
guanylate cyclase	4.6.1.2	NA
long-chain-fatty-acid—CoA ligase	6.2.1.3	NA
DNA ligase (ATP)	6.5.1.1	NA
DNA ligase (NAD+)	6.5.1.2	NA
cytochrome-c oxidase	7.1.1.9	NA
Flavodiiron (Flv1a/3a)	NA	NA

Table 3: The maximum growth rate, µ_max (d^-1) achieved for each O₂ experiment, for each strain, with the corresponding photoperiod, PAR level and spectral waveband.

Strain	Photoperiod (h)	PAR (µmol photons m-2 s-1)	Spectral waveband (nm)	[O2] (µM)	µmax (d-1)
MED4	16	180	450	250	0.68
MED4	12	90	450	25	0.65
MED4	All tested	All tested	All tested	2.5	0.00
SS120	16	90	White LED	250	0.50
SS120	8	90	450	25	0.45
SS120	12	30	660	2.5	0.15
MIT9313	16	30	450	250	0.54
MIT9313	16	90	White LED	25	1.01
MIT9313	12	90	450	2.5	0.45

Data availability

All data obtained from the Multicultivator were saved as comma separated values files and are available at https://github.com/FundyPhytoPhys/prochlorococcus_o2/Data/RawData. Annotated code for data import, transformations and analyses are available at https://github.com/FundyPhytoPhys/prochlorococcus_o2/Code.

XXX DOI pending from BOREALIS? or DRYAD? XXX

Acknowledgements

Carlie Barnhill (Mount Allison Student) assisted with code for import of multicultivator growth data files. Sarah Arthur (Mount Allison Student) assisted with culturing and setting up Multicultivator runs.

Funding

To Be Entered through PLoSONe system and deleted here Czech Academy of Science (OP) visiting fellowship supporting DAC work at AlgaTech Canada Research Chair in Phytoplankton Ecophysiology (DAC) Natural Sciences and Engineering Research Council of Canada, ‘Latitude and Light’ (DAC) Canada Foundation for Innovation (DAC) New Brunswick Foundation for Innovation (DAC) Rice Graduate Fellowship 2021 and 2022 (MS)

XXX PLOS requires grant numbers XXX

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